Project Summary The long term goal of our studies are focused on the elucidation of mechanisms by which metals cause or are associated with neurological disorders such as Alzheimer's disease (AD) and Wilson disease (WD). This experiments described in this proposal will focus on the role of copper in WD, a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. Although the hallmarks of WD, hepatic copper accumulation leading to fulminant hepatitis and/or cerebral copper accumulation leading to severe neurological symptoms, are known, the mechanisms by which accumulated copper triggers molecular changes at the organ or cellular levels is not very well understood. The central hypothesis of our proposal is that accumulating hepatic copper causes changes in the hepatocellular redox potential ultimately pushing the cells toward apoptosis or necrosis. We will test our hypothesis in 4 specific aims using a combination of spectroscopic approaches tailored to be used with mammalian tissue and traditional biochemical methods. Using synchrotron-based X-ray fluorescence in combination with a gold- based, dual labeling technique we will determine the cellular and intracellular copper concentration, distribution, and oxidation states in hepatic tissues of control mice and ATP7b-/- mice, an animal model for WD, at crucial disease stages (Specific Aim 1). We will then identify the intracellular sites (organelles) of copper accumulation and identify candidate proteins that could function as copper binding proteins in hepatic tissue of ATP7b-/- mice using fractionated homogenates and a combination of SXRF/fluorescence microscopy (Specific Aim2). Redox potentials in cellular models (HepG2 and MC65 cells) for the three central thiol/disulfide redox couples (GSH/GSSG, Trx1(SH)2/SS, Cys/CySS) will be determined under resting and high copper conditions (Specific Aim 3). The results will be subsequently compared to the redox potential of ATP7b-/- and control livers at different ages (Specific Aim 4). Correlation of our findings from Specific Aim 1 and 2 with those for Specific Aim 3 and 4 will allow us to characterize the effect of accumulating copper on the redox potential in cells and the subsequent changes in the cycle towards apoptosis or necrosis.